The present invention relates generally to the identification and isolation of a novel Shc-binding proteins, to novel nucleic acid molecules encoding such polypeptides and more particularly to the isolation and identification of a unique Shc binding protein designated PAL (Protein expressed in Activated Lymphocytes), and nucleic acid molecules encoding PAL.
The ubiquitously expressed Shc adapter proteins play a role in coupling growth factor receptor activation to intracellular signaling pathways. The mammalian Shc gene encodes at least three overlapping proteins with molecular weights of approximately 46 kDa, 52 kDa and 66 kDa (also called the p46, p52, and p66 isoforms or proteins, Bonfini et al., TlBS 21:257-261 (1996); Migliaccio et al., EMBO J. 16:706-716 (1997); Pelicci et al., Cell 70:93-104 (1992)). All three protein products share a carboxy terminal Src Homology 2 (SH2) domain, a central glycine/proline rich domain with homology to alphal collagen (CH1), and an amino terminal phosphotyrosine binding (PTB) domain which is different from the SH2 domain. The p52 and p46 isoforms differ only by 46 amino acids at the extreme amino terminus and are generated by the use of alternative translation initiation sites (Pelicci et al., Cell 70:93-104 (1992)). The p66 isoform is produced via alternative splicing of the Shc gene and contains an amino terminal extension which encodes a second collagen homology region (CH2) in addition to the common PTB, Ch1, and SH2 domains. Interestingly, it has recently been demonstrated that the expression of p66 is more restricted, and that some of its biological properties are distinct from those of the p52 and p46 Shc isoforms (Bonfini et al., TIBS 21:257-261 (1996); Migliaccio et al., EMBO J. 16:706-716 (1997)).
Shc proteins are typically tyrosine phosphorylated following activation of receptor tyrosine kinases (van der Geer et al, Ann. Rev. Cell Biol. 10:251-337 (1994)), such as the epidermal growth factor receptor (EGFR) (Pelicci et al., Cell 70:93-104 (1992)), the platelet-derived growth factor receptor (PDGFR) (Yokote et al., J. Biol. Chem. 269:15337-15343 (1994)), the nerve growth factor receptor (TrkA) (Obermeier et al., EMBO J. 13:1585-1590 (1994)), the insulin receptor (Pronk et al., J. Biol. Chem. 268:5748-5753 (1993)), and erbB-2 (Segatta et al., Oncogene 9:2105-2112 (1993)), as well as following activation of receptors that lack intrinsic tyrosine kinase activity, such as the T-cell receptor (TCR) (avichandran et al., Science 262:902-905 (1993)), the B-cell receptor (Saxton et al., J. Immunol. 153:623-636 (1994)), the receptors for the interleukins (Bums et al., J. Biol. Chem. 268:17659-17661 (1993); Cutler et al., J. Biol. Chem. 268:21463-21465 (1993); Ravichandran et al., J. Biol. Chem. 269:1599-1602 (1994)), and the erythropoietin receptor (Damen et al., Blood 82:2296-2303 (1993)). Additionally, tyrosine phosphorylation of Shc proteins has been detected after activation of G-protein coupled receptors (Cazaubon et al., J. Biol. Chem. 269:24805-24809 (1994); Chen et al., EMBO J. 15:1037-1044 (1996);al., Ptazniket et al. J. Biol. Chem. 270:19969-19973 (1995); Touhara et al., Proc. Natl. Acad Sci. USA. 92:9284-9287 (1995); van Biesen et al., Nature 376:781-784 (1995)), ligation of integrins (Maniero et al., EMBO J.. 14:4470-4481(1995); Wary et al., Cell 87:733-743 (1996)), and in cells expressing activated Src, Fps, Sea or Lck (Baldari et al., Oncogene 16:1141-1147 (1995); Crowe et al., Oncogene 9:537-544 (1994); McGlade et al., Proc. Natl. Acad. Sci. USA 89:8869-8873 (1992); Pelicci et al., Oncogene 11:899-907 (1995)), which implicates Shc proteins as important substrates of cytoplasmic tyrosine kinases.
Shc Protein Binding
Shc proteins are able to directly bind to tyrosine-phosphorylated peptides or proteins, including activated receptor tyrosine kdnases, typically by virtue of their SH2 or PTB domains (Bonfini et al., TIBS 21:257-261 (1996); Pawson, Nature 373:573-579 (1995)). The Shc SH2 domain preferentially binds to tyrosine phosphorylated peptides in the sequence context pY-E/I -X-I/L/M (where X represents any amino acid) (Songyang et al., Mol. Cell. Biol. 14:2777-2785 (1994)) and mediates the binding of Shc to the PDGFR (Songyang et al., Mol. Cell. Biol. 14:2777-2785 (1994)), EGFR (Pelicci et al, Cell 70:93-104 (1992)), receptor tyrosine kinase (RET) (Pronk et al. J. BioL Chem. 268:5748-5753 (1993)), and the CD3 zeta chain (Ravichandran etal., Science 262:902-905 (1993)). The PTB domain of Shc proteins also recognizes tyrosine phosphorylated peptides, but in a different context by selecting specific residues amino terminal to the phosphorylated tyrosine (Songyang et al., J. Biol Chem. 270:14863-14866 (1995)). The Shc PTB domain has been shown to bind directly to N-P-X-pY sequence motifs in the cytoplasmic domains of the EGFR (Blaikie et al., J. Boil Chem. 269:23031-32034 (1994)), TrkA (Diklic etal., J. Biol. Chem. 270:15125-15129 (1995)), RET (Lorenzo et al., Oncogene 14:763-771 (1997)), and the IL-2Rxcex2 chain (Ravichandran et al., Proc. Natl. Acad. Sci. USA 28:5275-5280 (1996)).
Phosphorylated Shc proteins are also able to associate with the Grb2 adapter protein by binding of the Grb2 SH2 domain to the phosphorylated tyrosine residue 317 (Y317) within the CH1 domain of Shc proteins (Rozakis-Adcock et al., Nature 360:689-692 (1992); Salcini et al., Oncogene 9:2827-2836 (1994)). Grb2 is stably associated with the Ras guanine nucleotide exchange factor, SOS (Batzer et al., Nature 363:85-88 (1993); Buday et al., Cell 73:611-620 (1993); Chardin et al., Science 260:1338-1343 (1993); Egan et al., Nature 363:45-51 (1993); Rozakis-Adcock et al., Nature 363:83-85 (1993)), and membrane localization of the Grb2-SOS complex results in activation of Ras (Aronheim et al., Cell 78:949-961 (1994)). Therefore, it has been proposed that Shc proteins are involved in coupling cell surface receptors to Ras activation. Several studies on the effects of Shc protein over expression provide support for this hypothesis. First, co-expression of a dominant negative mutant of Ras blocks neurite outgrowth in PC12 cells induced by Shc protein over expression (Rozakis-Adcock et al., Nature 360:689-692 (1992)). Second, over-expression of Shc protein in NIH 3T3 fibroblasts results in transformation (Pelicci et al., Cell 70:93-104 (1992)), and this can be abrogated by mutation of the presumed Grb2 binding site (Salcini et al., Oncogene 9:2827-2836 (1994)). Also, over-expression of Shc protein enhances EGF induced activation of MAP kinases (Migliaccio et al., EMBO J. 16:706-716 (1997)), and cell motility and growth in response to hepatocyte growth factor (HGF) (Pelicci et al., Oncogene 10:1631-1638 (1995)).
The modular structure of Shc proteins permits their interaction with multiple signaling molecules, suggesting that Shc proteins could finction to couple activated receptors to pathways other than Ras. Two additional sites of Shc protein tyrosine phosphorylation have recently been mapped to tyrosine residues 239 and 240 (Y239/240) (Gotoh et al., EMBO J. 15:6197-6204 (1996); Harmer et al., Mol. Cell. Biol. 17:4087-4095 (1997); van der Geer et al. Curr. Biol. 6:1435-1444 (1996)). Tyrosine 239 is present within a Grb-2 SH2 binding motif, and has been demonstrated to associate with Grb-2 in vivo (Gotoh et al., Mol. Cell. Biol. 17:1824-1831 (1997); Harmer et al. Mol. Cell. Biol. 17:4087-4095 (1997)). The Y239/240 phosphorylation sites may also couple Shc proteins to additional downstream SH2 containing proteins, since phosphopeptides corresponding to Y239/240 have been demonstrated to bind to a variety of as yet, unidentified proteins, in addition to Grb2 (van der Geer et al., Curr. Biol. 6:1435-1444 (1996)). A novel role for Shc proteins has been suggested in which phosphorylation of Y239/ Y240 leads to c-myc induction, and suppression of apoptosis in Ba/F3 cells, in a Ras-independent manner (Gotolet al., EMBO J. 15:6197-6204 (1996)).
The Shc PTB domain has been demonstrated to bind directly to the cytoplasmic enzyme SHIP, an SH2 domain containing inositol 5-phosphatase, in response to growth factor and cytokine stimulation in hematopoietic cells (Damen et al., Proc. Natl. Acad Sci. USA 93:1689-1693 (1996); Kavanaugh et al., Curr. Biol. 6:438-445 (1996); Lioubin et al., Genes and Development 10:1084-1095 (1996)). Furthermore, proline rich sequences in the Shc CH1 domain are proposed to mediate the interaction between Shc proteins and the SH3 domain of eps8, a tyrosine phosphorylated protein involved in EGF receptor mediated signaling (Bonfini et al., TIBS 21:257-261 (1996); Matoskova et al., Mol. Cell. Biol. 15:3805-3812 (1995)). Therefore, it is likely that Shc proteins participate in diverse signal transduction pathways by interacting with multiple cytoplasmic signaling molecules. Shc proteins are generally believed to be involved in various cell proliferation pathways. Specifically, Shc proteins appear to be involved in signaling pathways that lead to cell division.
The invention is directed to an isolated and purified Shc binding protein designated PAL (Protein expressed in Activated Lymphocytes ) and to nucleic acids encoding the proteins.
In certain embodiments, the invention is directed to a PAL polypeptide comprising the polypeptide set out as SEQ ID NO.:2; the polypeptide set out as SEQ ID NO.:4; a polypeptide that is at least 75 percent identical to the foregoing polypeptides; and a biologically active fragment, homolog or variants, conserved variants, allelic variant analogs. The PAL polypeptide optionally may have an amino terminal methionine. The polypeptides of the invention may also be covalently modified with for example water soluble polymers, and fusion with peptides preferably at the amino or carboxy terminus of a PAL polypeptide according to the invention. Also encompassed by the invention are polypeptides having about 50% sequence similarity to the polypeptides set out as SEQ ID NOS.:2 or 4.
The invention is further directed to anti-PAL antibodies directed against such PAL polypeptides.
In certain embodiments, the present invention is directed to a polynucleotide molecule encoding a polypeptide selected from the group comprising of the polynucleotide molecule of SEQ ID NO.:1; the polynucleotide molecule of SEQ ID NO.:3; a polynucleotide molecule encoding the polypeptide of SEQ ID NO.:2 or a biologically active fragment thereof; a polynucleotide molecule that encodes a polypeptide that is at least 75 percent identical to the polypeptide of SEQ ID NO.:2; a polynucleotide molecule encoding the polypeptide of SEQ ID NO.:4 or a biologically active fragment thereof; a polynucleotide molecule that encodes a polypeptide that is at least 75 percent identical to the polypeptide of SEQ ID NO.:4; a polynucleotide molecule that hybridizes under stringent conditions to a polynucleotide molecule complementary to any of items (a)-(f) below; and a polynucleotide molecule that is the complement of any of items (a)-(g) below.
In other embodiments, the invention is directed to vectors comprising these polynucleotide molecules, and host cells, either prokaryotic or eukaryotic, comprising the vectors, and recombinant host cells containing the polynucleotide molecules or vectors according to the invention.
In other embodiments, the invention is directed to a polynucleotide or fragment(s) thereof, which can be used to detect the presence of polynucleotide molecules that encode PAL polypeptides. In other embodiments, the invention provides methods of using such nucleic acids or fragments to detect the presence of nucleic acids encoding PAL polypeptides.
In other embodiments, the invention provides a process for producing a PAL polypeptide including the polypeptides described above.
In yet other embodiments, the invention is directed to a process for producing a recombinant host cell that expresses a PAL polypeptide according to the invention, and to methods of producing PAL polypeptides using those host cells.
In still another embodiment, the invention is directed to a mammalian cell containing a PAL polypeptide encoding DNA and modified in vitro to permit higher expression of PAL polypeptide by means of a homologous recombinational event consistent of inserting an expression regulatory sequence in functional proximity to the PAL encoding DNA thereby increasing the expression of a PAL polypeptide by activating of an endogenous PAL gene.
In other embodiments, the invention provides a process of using such recombinant host cells to screen compounds or compositions for their ability to block cell division or proliferation.
In yet other embodiments, the invention provides a transgenic animal that produces PAL polypeptide, according to the present invention on to transgenic animals in which one or more of its PAL genes is disrupted or xe2x80x9cknocked outxe2x80x9d and
The invention also provides a process of using such a transgenic animal to screen compounds or compositions for their ability to block cell division or proliferation by measuring the effect on PAL production and inhibition of cell proliferation in vivo in the transgenic animal by the compounds or compositions.